PROJECT SUMMARY: The ?Functional and Transcriptomic Atlas of CF Respiratory Epithelial Cells? seeks to establish a Core to isolate and expand respiratory epithelial cells from both nasal and bronchial brushings from patients with specific CFTR mutations. Cells will be cultured in bronchospheres and nasospheres. Fluid and electrolyte secretion will be assessed in the cultures. Effects of CFTR mutations on cell-specific transcriptomes will be studied at single cell level. Confocal microscopy and immunofluorescence will be used to carefully identify the effects of CFTR mutations and ?correctors? on cell differentiation, phenotypes, and functions. Detailed cell-specific RNA databases will be produced for nasal and bronchial cell cultures using single cell RNA-seq. Detailed molecular imaging, by confocal microscopy, will be used to create an Atlas of gene expression and behavior related to CF. Effects of CFTR correctors on specific patient mutations will be explored in these in vitro models. Specific Aim 1 will isolate, culture, and evaluate respiratory epithelial cells for study of CF from bronchial brushings. Specific Aim 2 will use NexGen single cell RNA-sequencing to create an Atlas of patient-specific respiratory epithelial cell transcriptomics. These studies will identify the effects of CFTR mutations on gene expression at the single cell level, identifying the effects of CFTR mutations and correctors on the behaviors of individual cells of multiple cell types, including ciliated, serous, and goblet cells that will be present in these cultures. The Core will create a molecular Atlas which will be cell-, patient-, and mutation-specific and will provide an extensive data set for use by investigators active in the CF research community worldwide. Such data will enable the ability to understand the pleotropic effects of CFTR, CFTR mutations, and CFTR correctors in a patient-specific manner, as we move toward patient-specific therapies for cystic fibrosis.